Evaluation of Intestinal Microbiota in Children With Sickle Cell Disease.

BACKGROUND AND AIMS: Sickle cell disease (SCD) is a chronic hemolytic anemia that may be life-threatening due to multisystemic effects. Identification of the factors which affect the pathophysiology of the disease is important in reducing mortality and morbidity. This study aimed to determine gut microbial diversity in children and adolescents with SCA compared with healthy volunteers and to evaluate the clinical impact of microbiota. MATERIALS AND METHODS: The study included 34 children and young adolescents with SCD and 41 healthy volunteer participants. The microbiome was assessed by 16S rRNA sequencing in stool samples. Laboratory parameters of all participants, such as complete blood count and C-reactive protein values and clinical characteristics of SCD patients, were determined and compared, as well as clinical conditions of the patients, such as vascular occlusive crisis and/or acute chest syndrome, frequency of transfusions, intake of penicillin, hydroxyurea, and chelation therapy were recorded. RESULTS: White blood cell count, hemoglobin, immature granulocyte and C-reactive protein levels were significantly higher in the patient group ( P <0.05). Microbiota analysis revealed 3 different clusters among subjects; controls and 2 clusters in the SCD patients (patient G1 and G2 groups). Bacteroides spp. were more prevalent, while Dialester spp. and Prevotella spp. were less prevalent in SCD compared with controls ( t =2.142, P <0.05). Patient G2 (n=9) had a higher prevalence of Bacteroides and a lower prevalence of Prevotella than patient G1 (n=25). CONCLUSION: In our study, there was a difference between SCD patients and the control group, while 2 different microbiota profiles were encountered in SCD patients. This difference between the microbiota of the patients was not found to affect the clinical picture (such as vascular occlusive crisis, acute chest syndrome).

[Experience of the National HIV-AIDS Reference Center of Turkey, in the Transition to the New HIV Diagnostic Algorithm; Comparative Analysis of Line-Immunoassay Test and Bio-Rad Geenius™ HIV-1/2 Antibody Confirmatory Assay]. / Yeni HIV Tani Algoritmasina Geçis Sürecinde Ulusal HIV-AIDS Referans Merkezi'nin Deneyimi: Line-Immunoassay Test ve Bio-Rad Geenius™ HIV-1/2 Antikor Ayirt Edici Hizli Dogrulama Testleri Karsilastirmali Analizi.

Shortly after the first detection of human immundeficiency virus (HIV) infection in USA in 1981, the number of cases have increased gradually from all around the world. Turkey's high capacity for tourism and the unique geographic location extending between Europe and Asia, provides convenience for the passage of individuals across the countries and sexually transmitted infections including HIV, as well. According to the official data of the Ministry of Health; there are 25809 HIV positive and 1958 AIDS cases as of November 30, 2020, after the epidemic started in 1985 in Turkey. Despite the decrease in the number of newly detected HIV cases as a result of serious measures taken for the transmission of infection worldwide, the increase in the number of cases still continues in our country. Shortening the reporting period and starting treatment as soon as possible in the diagnosis of infection is critical for the control of the epidemic. For this purpose, Centers for Disease Control and Prevention (CDC) published a new test algorithm in 2010, which suggested the use of the Geenius™ HIV ½ supplemental assay test instead of western blot tests, which have been used for many years to verify HIV screening test positivity. In this study, we aimed to report the experience of the National HIV-Acquiner Immundeficiency Syndrome(AIDS) and Viral Hepatitis Reference Laboratories of Turkey in the first year of transition to the new HIV algorithm and to evaluate the diagnostic performance of Geenius™ HIV ó and line immunassay (LIA) s. A total of 2090 anti-HIV positive patient sera sent to National HIV-AIDS and Viral Hepatitis Reference Laboratories of Turkey, Ankara for HIV confirmation were included in the study. All samples were retested with a fourth-generation enzyme linked immunosorbent assay (ELISA) test (VIDAS® HIV-1/2 Duo Ultra assay, BioMerieux, France) followed by the confirmatory tests; Geenius™ HIV 1/2 confirmatory assay (BioRad, Redmond, WA) and Line-immunoassay (INNO-LIA HIV ½ Score, Fujirebio, reverse transcriptase polymerase chain reaction (RT-PCR) (artus HI Virus-1 RT-PCR, Qiagen, Germany) test and in-house HIV-2 RNA and proviral DNA PCR. The sensitivity, specificity, and the agreement of the each assay were compared. Cohen's Kappa analysis was used for the evaluation of the agreement between the tests. According to the new algorithm which recommended Geenius™ test besides HIV-1 RNA test, 1707 (81.7%) HIV-1 positive samples were identified. Of these samples; 95.9% and 95.02% were identified as HIV-1 positive by GeeniusTM and INNO-LIA, respectively. However, 2.5% of the positive samples were negative with Geenius™ and 3.5% with INNO-LIA. One and a half percentage (1.5%) of these samples were detected with Geenius™ and 1.4% with INNO-LIA as indeterminant. When all the positive samples determined with ELISA were evaluated; it was detected that,1.3% were indeterminate by Geenius™ test and 2.4% by the INNO-LIA test. When the INNO-LIA test was regarded as the gold standard method; sensitivity, specificity, positive predictive and negative predictive values of the Geenius™ test were as follows; 99.7%, 96.1%, 98.9%, and 99.1%. The agreement between INNO-LIA and Geenius™ tests was found to be 98.95% (κ= 0.969; very good). When the Geenius™ and HIV-1 PCR tests were evaluated together for the confirmation; the sensitivities of Geenius™ and INNO-LIA tests were 99.8% and 98.3%, specificities were 89.8% and 85.3%, respectively. Slight positive bands were detected in the gp36 or gp140 bands, the HIV-2 specific envelope proteins, were detected in seven samples, However, the positivity disappeared after the dilution of the samples and it was accepted as false positivite reaction due to the absence of HIV-2 RNA and proviral DNA in these samples. In conclusion; we concluded that Geenius ™ and INNO-LIA tests have a perfect agreement in HIV diagnosis and due to the rapid and reliable results provided for the HIV test protocol, Geenius™ test can be used safely as an alternative to the immunoblot tests. HIV-1 RNA testing must be performed in all HIV confirmation centers in order to detect acute HIV cases in the fast and early period which are the main reason for the updates in HIV diagnosis.

[A Case of Chronic Q Fever Endocarditis Mimicking Lymphoproliferative Disorders]. / Lenfoproliferatif Hastaliklarla Karisan Bir Kronik Q Atesi Endokarditi Olgusu.

Q fever is a zoonosis caused by Coxiella burnetii. In this report, a case of chronic Q fever endocarditis with pancytopenia and hypergammaglobulinemia mimicking a lymphoproliferative disease was presented. A 39-years-old male living in Çatalca and whose family is engaged in animal husbandry admitted with the complaints of weakness and fatigue. The patient had aortic valve replacement 29 years ago and had aortic valve re-replacement, and ascending aorta grafting because of endocarditis three years ago. It was revealed that the second operation of the patient was due to possible infective endocarditis, but no definitive agent could be identified. He was evaluated for massive hepatosplenomegaly, pancytopenia, hypergammaglobulinemia, presence of M-spike and elevated ß-2 microglobulin levels and was referred to our hematology clinic with a preliminary diagnosis of lymphoproliferative disease. Lymphoplasmacytic lymphoma was excluded with the result of bone marrow biopsy and he was referred to our clinic for the investigation of possible infectious etiologies. We detected hepatosplenomegaly and finger clubbing. His blood analyses were normal except for the following: leukocyte count 3800/µl, platelet count 148000/µl, gamma globulin 5.9 gr/dl, rheumatoid factor (RF) and antinuclear antibody (ANA) positivity. Chronic Q fever endocarditis was suspected and C.burnetii Phase I IgG test was found positive in 1/132071 titers. Although transesophageal echocardiography showed no lesion of endocarditis, positron emission tomography/computed tomography revealed increased fluorodeoxyglucose uptake around the prosthetic heart valve and graft. The patient was diagnosed as having Q fever endocarditis and graft infection. He refused hospitalization and was started on hydroxychloroquine and doxycycline treatment. The patient stopped taking these antibiotics by himself seven days after the diagnosis. He was admitted with a headache to another hospital and operated for an intracranial hemorrhage and died shortly after. Apart from unfamiliarity, wide range of clinical presentations of disease could also lead to delayed diagnosis. Among patients with chronic Q fever, continuous bacteremia and antigenic stimulus causes inflammatory syndrome with hepatosplenomegaly, hypergammaglobulinemia and, presence of autoantibodies which leads to misdiagnoses of rheumatologic, autoimmune or hematologic diseases Chronic Q fever should be investigated in patients with known valvulopathy and chronic hepatomegaly or splenomegaly, pancytopenia, hypergammaglobulinemia, and unexplained autoantibody positivity.

[The Epidemiology of Carbapenemases in Escherichia coli and Klebsiella pneumoniae Isolated in 2019 in Turkey]. / Türkiye'de 2019 Yili Içinde Izole Edilen Escherichia coli ve Klebsiella pneumoniae Izolatlarinda Karbapenemaz Epidemiyolojisi.

Antibiotic resistance is one of the most important public health problem and one of the most critical steps in preventing resistance is the monitorization of the resistance. Local, regional and global monitoring enables the spread of antibiotic resistance to be understood more clearly. In this study, it was aimed to evaluate the results of the pilot study for the establishment of molecular-based carbapenem surveillance system in Escherichia coli and Klebsiella pneumoniae isolates and to investigate the carbapenemase epidemiology in Turkey. Hospitals (n= 28) from 26 different statistical level II regions from Turkey were included in the study. The hospitals participated in the study submitted ten carbapenem susceptible and ten carbapenem resistant E.coli and K.pneumoniae isolates to our laboratory that were isolated in two different periiods of six-month either between 1 March-31 August or 1 April-30 September 2019. A total of 509 isolates were collected from 26 of the 28 participating hospitals in the study. Isolates were identified by matrix assisted laser desorptionization-time of flight mass spectrophotometry (MALDI TOF MS) (Bruker Daltonics, Germany) method and antibiotic susceptibility tests for imipenem, meropenem and colistin were studied by broth microdilution. Moreover, susceptibilities to amikacin, amoxicillin-clavulanic acid, ampicillin, aztreonam, cefepime, cefotaxime, ceftazidime, ciprofloxacin, ertapenem, gentamicin, piperacillin-tazobactam, tobramycin and trimethoprim-sulfamethoxazole were determined by disc diffusion method. The resistance genes were investigated in isolates which were found to be phenotypically resistant to carbapenem and colistin, in house method was used to investigate carbapenemase genes and a commercial colistin resistant real-time PCR kit (Biospeedy, Turkey) was used for colistin resistance genes. In total, 493 of the 509 isolates collected from hospitals were identified as E.coli (25.7%, n= 127) and K.pneumoniae (74.3%, n= 366) and included in the study. It was determined that 31% of the isolates evaluated were from community-acquired infections and 69% were either from healthcare-associated infections or from colonization sites. Among the tested isolates, 248 (50.3%) were susceptible to carbapenems and 245 (49.7%) were resistant. The types of carbapenemases in carbapenemase-producing were OXA-48 (52.2%), KPC (16.1%), NDM-1 (15%), OXA-48 + NDM-1 (12.6%), KPC + NDM-1 (2.8%) and VIM (0.5%) and OXA-48+VIM (0.5%). Resistance to colistin was detected in 23.3% of the isolates but mcr1-8 genes were not detected. It was found that all colistin resistant isolates are resistant to at least one of the carbapenems. The importance of a molecular-based antimicrobial resistance surveillance system in our country was demonstrated with this pilot study. It is thought that continuous monitoring of these epidemiological features will contribute to the management of infections due to carbapenemase-producing organisms.

[Determination of the Subspecies of Francisella tularensis Isolated in Turkey by Molecular Methods]. / Türkiye'de Izole Edilen Francisella tularensis Alt Türlerinin Moleküler Yöntemlerle Belirlenmesi.

Francisella tularensis is a gram-negative, coccobasillus, facultative intracellular bacteria and causes a zoonotic disease, tularemia in humans. F.tularensis has four subspecies, which have different virulences for humans as F.tularensis subsp. tularensis, F.tularensis subsp. holarctica, F.tularensis subsp. mediasiatica and F.tularensis subsp. novicida. F.tularensis subsp. tularensis is the most virulent subspecies and mortality rate is high in human cases. F.tularensis subsp. holarctica, which has been reported in our country to date, has lower virulence than that of subsp. tularensis, and causes rare lethality among untreated patients. According to the erythromycin resistance and the properties of glucose-glycerol fermentation, F.tularensis subsp. holarctica has three biovar as biovar I, biovar II and biovar japonica. F.tularensis subsp. mediasiatica has been reported only in a few central asian countries and its virulence is similar to the F.tularensis subsp. holarctica F.tularensis subsp. novicida is avirulent for immunocompetent individuals but has been observed to cause infection in immunocompromised individuals. The aim of this study was to determine the F.tularensis subspecies in 259 F.tularensis strains isolated from clinical specimens, drinking water and a rodent sample and 517 F.tularensis PCR-positive DNA isolated from clinical specimens between years 2009 and 2014. Conventional PCR was performed using primers specific for the RD1 (Region Difference) region of F.tularensis. Subspecies were differentiated depending on the difference in PCR amplification product size. In our study, F.tularensis subsp. holarctica was detected in 764 samples yielding 922 base pair (bp) amplification product. The DNA samples obtained from one water and 11 lymph aspirates were determined as F.tularensis subsp. holarctica biovar japonica. The DNA sequence analysis of the amplification product of the RD1 region of the isolate from water sample was determined. The 1136 bp nucleotide sequence obtained from the DNA sequence analysis was 100% similar to F.tularensis subsp. holarctica biovar japonica (FCS075 strain-accesion number AF469618) when compared with GenBank data. The whole genome sequence of this isolate was also determined and recorded to GenBank with accesion number CP007148. None of the samples used in our study belonged to other sub-species. F.tularensis subsp. holarctica biovar japonica positive 11 lymph aspirate samples were sent to our center from Ankara (n= 1), Kayseri (n= 1) and Afyon (n= 9) provinces. The results of the current study revealed that F.tularensis subsp. holarctica biovar japonica caused a tularemia outbreak in a village in Afyon province at first time and it was observed sporadically in two other different provinces.

[Direct Identification of Microorganisms from Positive Blood Cultures by MALDI-TOF MS Using an in-House Tween® 80 Method: Experimental and Clinical Study]. / "In-House" Tween® 80 Yöntemi ile Pozitif Kan Kültürlerinden Mikroorganizmalarin MALDI-TOF MS Yöntemi ile Dogrudan Tanimlanmasi: Deneysel ve Klinik Çalisma.

It has been reported that direct identification from blood culture bottles with positive signals and reporting the results to the clinics earlier has positive effects on mortality and morbidity. Extraction methods especially using detergents are used for the direct identification from the bottles which give positive signal. For this purpose, in-house methods developed based on the usage of saponin are widely available in the literature. In this study, it was aimed to develop a simple, easy-to-apply and reliable protocol for identifying the agent directly from the blood culture bottle that gives positive signal with the use of detergent Tween® 80, and to study the obtained protocol in clinical samples in a routine microbiology laboratory and to evaluate the results. The study was carried out in two stages, the experimental stage where the method was developed and the clinical stage where the method was applied. In the experimental stage, blood culture bottles were created with standard strains and isolates previously diagnosed with the 16S rRNA method. 10% solution of Tween® 80 was prepared with distilled water. 1 ml sample was transferred from the bottle that gave positive signal to the microcentrifuge tube, 100 µl of 10% solution of Tween® 80 was added, vortexed for 10 seconds and then incubated for 5 minutes at room temperature. The tubes were centrifuged for 5 min at 14.000 rpm, the supernatant was discarded and the pellet was washed with 1 ml of distilled water and centrifuged at 14.000 rpm for 5 minutes in three times. Samples taken from the pellets were rubbed on the slide and dried on air. Firstly, 1 µl of 70% formic acid, then 1 µl, of matrix solution was added and it was used after drying. In the second stage of the study, the method was applied to the 502 vials giving positive signal in the Microbiology Laboratory of Ankara University Faculty of Medicine Ibni Sina Hospital between 17 April 2018-31 August 2018 and the results were compared with the subculture results. The results obtained at the end of extraction in the experimental stage were compared with the subculture results and no statistical difference was found. In 383 (82.9%) bottles among 462 (92.1%) bottles with monomicrobial positive cultures, compatible results with the subculture results were obtained. Of the microorganisms correctly identified, 350 (91.3%) were bacteria and 33 (8.7%) were fungi. On the other hand, 216 (56.4%) of the bacteria were gram positive and 134 (34.9%) of them were gram negative bacteria. At least one microorganism was correctly identified in 19 (47.5%) of 40 (7.9%) bottles with polymicrobial blood cultures. Their distribution was gram negative (n= 10) and gram positive (n= 8) and yeast (n= 1). No microorganisms were identified in six bottles with polymicrobial cultures. According to the results, we believe that this in-house method developed using Tween® 80 will be a routinely applicable method for blood culture bottles that give positive signal in microbiology laboratories and it will contribute to the early diagnosis.

Coronaviruses and SARS-COV-2

Coronaviruses (CoVs) cause a broad spectrum of diseases in domestic and wild animals, poultry, and rodents, ranging from mild to severe enteric, respiratory, and systemic disease, and also cause the common cold or pneumonia in humans. Seven coronavirus species are known to cause human infection, 4 of which, HCoV 229E, HCoV NL63, HCoV HKU1 and HCoV OC43, typically cause cold symptoms in immunocompetent individuals. The others namely SARS-CoV (severe acute respiratory syndrome coronavirus), MERS-CoV (Middle East respiratory syndrome coronavirus) were zoonotic in origin and cause severe respiratory illness and fatalities. On 31 December 2019, the existence of patients with pneumonia of an unknown aetiology was reported to WHO by the national authorities in China. This virus was officially identified by the coronavirus study group as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the present outbreak of a coronavirus-associated acute respiratory disease was labelled coronavirus disease 19 (COVID-19). COVID-19's first cases were seen in Turkey on March 10, 2020 and was number 47,029 cases and 1006 deaths after 1 month. Infections with SARS-CoV-2 are now widespread, and as of 10 April 2020, 1,727,602 cases have been confirmed in more than 210 countries, with 105,728 deaths.

[Isolation of Rickettsia slovaca by shell-vial cell culture method from Dermacentor marginatus ticks]. / Shell-vial hücre kültürü yöntemi ile Dermacentor marginatus türü kenelerden Rickettsia slovaca izolasyonu.

Rickettsia species are gram negative, small pleomorphic coccobacilli, obligate intracellular bacteria. The majority of these bacteria are transported by the ticks. Rickettsia slovaca and Rickettsia raoultii are among the Rickettsia species in the spotted fever group and carried by Dermacentor species ticks, that cause tick-borne lymphadenopathy (Tickborne Lymphadenopathy-TIBOLA or Dermacentor-borne necrotic erythema and lymphadenopathy- DEBONEL). Rickettsia species are obligate intracellular bacteria and they can be cultivated in cell cultures. The chance of isolation of Rickettsia species from clinical and tick specimens has been increased by the shell-vial centrifugation cell culture method. The aim of this study was to isolate R.slovaca from two Dermacentor marginatus species ticks which were detected in humans by the use of shell-vial centrifugation cell culture method using VERO cell. Before proceeding to the culture stage, an algorithm including description, disinfection, dissection of ticks and methods of hemolymph, homogenization, DNA extraction and real-time PCR was performed. Iodine-alcohol disinfection was performed following identification of the ticks with the use of a stereo microscope. Ticks hemolymph were obtained with extremity dissection of ticks and indirect fluorescence antibody (IFA) test was performed with Rickettsia positive sera. Ticks identified as containing Rickettsia like bacteria in hemolymph were homogenized and DNA extraction was performed from homogenate of ticks. By real-time PCR, using Rickettsia genus-specific PanR8 primers and probes, PCR positive Rickettsia spp. were determined among the ticks. Vero cells that have formed monolayer in vials were used for Rickettsia culture. Homogenized tick samples which were positive for Rickettsia in hemolymph and real-time PCR methods were inoculated to cell culture vials. Suspended bacteria in the inoculum were allowed to approach the cells by the shell-vial centrifugation method. Cells were scraped after five days of incubation in 5% CO2 at 36°C. An aliquot of the determined cell suspension was fixed to the slides and then IFA was performed using Rickettsia positive sera. In fluorescence microscopy examination, adhered and proliferated bacteria were observed on Vero cells. This cell suspension was again inoculated into the Vero cell cultures to increase bacterial replication and taken for 5-7 days of incubation. DNA was extracted from the suspension of the cultivated bacteria. Conventional PCR of citrate synthase (gltA) and outer membrane protein A (ompA) gene regions were used to identify Rickettsia species. DNA sequence analysis of PCR amplification products were determined. DNA sequence results were compared to Genbank data and found that the gltA sequence was 100%, similar to R.slovaca with accession number AY129301.1 and the ompA sequence was 100%, similar to R.slovaca with accession number KF791242.1.  In addition, the two strains isolated in phylogenetic analysis were found to be R.slovaca. As a result, R.slovaca isolation from of D.marginatus type ticks has been reported for the first time in our country by the cell culture method.

Prevalence and diversity of rotavirus A genotypes cirulating in Turkey during a 2-year sentinel surveillance period, 2014-2016.

Human rotavirus A (RVA) is the main etiological agent of watery diarrhea among children under 5 years of age worldwide. The aims of this study were to investigate the prevalence and diversity of RVA genotypes circulating in Turkey during a 2-year sentinel surveillance study. A total of 1639 rotavirus antigen-positive stool samples were obtained from children younger than 5 years of age hospitalized with acute gastroenteritis. Rotavirus G and P genotypes were determined by reverse transcription polymerase chain reaction (RT-PCR) with consensus primers for the VP7 and VP4 genes, followed by semi-nested type-specific multiplex PCR. Rotavirus RNA was detected in 1396 (85.3%) of the samples tested. The highest detection rate (38.2%) was obtained among children in the 0-12 months age group, followed by children in the 13-24 months age group (36.2%). The most prevalent genotype was G1P[8] (24.6%) followed by G3P[8] (19.6%), G9P[8] (12.2%), G2P[4] (9.5%), G2P[8] (6.5%), and G4P[8] (4.8%). The proportions of uncommon and mixed genotypes were 21.5% and 1.14%, respectively. The large number of genotypes observed, including common, uncommon, and mixed types, indicates a high heterogeneity of RVA strains circulating in Turkey. The current study also exhibited dramatic fluctuations on the prevalences of the common genotypes, with increases in G3 and G1 and decreases in G9 and G2 from 2014-2016.

[A Rickettsia case caused by Rickettsia conorii]. / Rickettsia conorii'nin neden oldugu riketsiyal enfeksiyon olgusu.

Rickettsia species are gram-negative intracellular, small pleomorphic coccobacilli in the Rickettsiaceae family. This genus is serologically and genotypically divided into four groups as spotted fever group, typhus group, Rickettsia belli and Rickettsia canadensis. Rickettsia conorii (R.conorii subsp. conorii) in the spotted fever group was reported to cause mediterranean spotted fever in Europe, especially in mediterranean countries including Turkey. The major vectors of Rickettsia species are ticks, and in some species fleas or mites. In this report a case with R.conorii infection was presented. A 46-year-old female patient, who had anorexia, fatigue, muscle aches, chills and high fever was admitted to a health institution. The patient was diagnosed as influenza. There was no regression in the patient's complaints with the recommended treatment. The patient was examined in our infectious diseases clinic and had several symptoms like severe muscle and joint pain with significant headache, and rashes at her body including hands and feet. The patient had a single eschar in the upper midline of the belly that matched tick biting and pink small maculopapular scars on the trunk, arms, legs, feet, and hands. Considering a Rickettsia pre-diagnosis, liquid electrolyte and doxycycline 2 x 100 mg oral treatment was started. On the third day of treatment, high fever, muscle and joint pain were decreased. On the fifth day, active skin lesions were started to fade. R.conorii IgM and IgG were negative in the first serum sample of the patient. In the biopsy sample taken from eschar tissue, Rickettsia spp. was detected as positive with rt-PCR. PCR was used by using the specific regions of the genetically specific gltA and ompA genes in the biopsy specimens and then the PCR products were determined by DNA sequence analysis. The DNA sequence results were compA red with Genbank data and determined that the gltA sequence was 99%, similar to R.conorii with accession number JN182786 and the ompA sequence was 99%, similar to R.conorii with accession number KR401144. When the phylogenetic tree was created, it was observed that the etiological agent was R.conorii. A week after the treatment, in the second serum sample R.conorii IFA IgM 1/192 titer and IgG 1/320 titer were detected as positive. In this case report, we have presented a Rickettsia case, clinically diagnosed as Rickettsia, serologically negative in the acute phase, PCR positive, with post-treatment seroconversion and etiologic agent determined as R.conorii.

Investigation of syphilis coinfection and performance of the Architect Syphilis Tp ELISA screening test in HIV positive patients

Background/aim: Limited data on syphilis coinfection in human immunodeficiency virus (HIV) positive cases exist in Turkey. Our aim is to investigate syphilis coinfection and to evaluate the compatibility of the screening Architect Syphilis Tp ELISA with the fluorescent treponemal antibody absorption (FTA-abs) confirmation test in HIV positive cases. Materials and methods: Totally 519 HIV positive patients were included in the study. Enzyme linked fluorescent assay (ELFA) was used as a screening test and positive samples were confirmed by line immunassay (LIA). In order to discriminate acute HIV infection and false ELISA positivity, HIV-1 RNA PCR was performed in ELFA positive and LIA negative samples. Architect Syphilis TP ELISA was used for the detection of total antibodies against Treponema pallidum in HIV positive patients. Positive results were confirmed by the FTA-abs test. Results: Out of 519 HIV-1 positive patients, IgG and IgM positivity, and only IgG positivity was detected as 1.9% and 11.4% in all the samples, respectively. A total of 79 (15.2%) sera were positive with Architect Syphilis Tp ELISA test and 69 (13.3%) were positive with FTA-abs test. Statistically significant, almost perfect agreement was found between Architect Syphilis Tp ELISA and FTA-abs tests (kappa = 0.921 and P < 0.001). Conclusion: Implementation of syphilis and HIV screening tests together among risk groups is considered to be appropriate.

Giant Orf on the Nose.

Orf is a zoonotic infectious disease caused by parapoxvirus. Orf lesions are typically seen on the hand, but they have rarely been reported on the nose. Herein, the authors report a rare patient of an orf lesion on the nose of a 52-year-old man after the Muslim celebration of the feast of the sacrifice. The lesion spontaneously recovered 8 weeks after the initial appearance and showed no evidence of recurrence after 1 year of follow-up. Orf virus infections may occur more often after the celebration of the feast of the sacrifice in Muslim countries.

Evaluation of clinical and serological findings for diagnosis of cutaneous anthrax infection after an outbreak.

PURPOSE: Anthrax, caused by the bacterium Bacillus anthracis, is one of the oldest documented infectious diseases in both livestock and humans. We aimed to evaluate clinical findings and risk factors of patients with cutaneous anthrax infection and report anti-lethal factor (LF) IgG and anti-protective antigen (PA) IgG titers in the serologic diagnosis of disease. METHODS: In this study, serum samples of 18 cutaneous anthrax patients were collected and anti-LF IgG and anti-PA IgG titers were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Twelve (67%) males and 6 (33%) females, with a mean age of 36.06 ± 16.58 years were included in the study. Risk factors identified in the patient population studied were slaughtering (28%), flaying (56%), chopping meat (67%), burying diseased animal corpses (17%) and milking (6%) livestock. Black eschar formation (94%), pruritus (78%) and painful lymphadenopathy (61%) were first three common clinical signs and symptoms, respectively. Fourteen (78%) patients produced a positive IgG response against PA, 11 (61%) patients produced against LF. Three (17%) patients had no response to either antigen. CONCLUSIONS: A detailed history of contact with sick animals or animal products along with clinical findings should be taken at the first step for the diagnosis of cutaneous anthrax infection. Serologic detection of anti-LF IgG and anti-PA IgG with ELISA may be useful auxillary method for establishing the diagnosis.

[Screening of Corynebacterium diphtheriae, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis in throat swab specimens of children with upper respiratory tract infections]. / Üst solunum yolu enfeksiyonu bulgulari ile basvuran çocuklarin bogaz sürüntü örneklerinde Corynebacterium diphtheriae, Corynebacterium ulcerans ve Corynebacterium pseudotuberculosis suslarinin arastirilmasi.

Although a significant decrease has been reported in the incidence of diphteria in many regions of the world following the routine diphtheria immunization programs, the emergence of new cases indicated that toxigenic strains are still circulating in the community. Diphtheria vaccine does not provide protection against asymptomatic carriage and colonization of non-toxigenic Corynebacterium diphtheriae. It is a known fact that invasive infections may arise from non-toxigenic C.diphtheriae strains that the non-toxigenic strains can become toxigenic strains leading to diphteria. It is also known that there is a risk of diphteria outbreaks due to decreased antitoxin level and inadequate adult immunization programs. In our country, there is no routine surveillance of toxigenic and non-toxigenic C.diphtheriae. In the present study we aimed to investigate the presence of C.diphtheriae, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis in children presenting with the symptoms of upper respiratory tract infections that might be confused with those moderate diphteria, in order to highlight the requirement of microbiological surveillance and to create awareness about these microorganisms among public health experts, microbiologists and clinicians. Throat swab specimens were obtained from children who were admitted to the pediatric outpatient clinics, in Dr. Sami Ulus Obstectrics, Children Health and Diseases Educational and Research Hospital, with upper respiratory tract infections between 1 February 2016-22 March 2016. The specimens were inoculated in 5% sheep blood agar plates. The plates that were incubated in appropriate conditions, were evaluated for Group A beta hemolytic streptococcocci. Subsequently, culture plates were sent to the Public Health Institution of Turkey, National Respiratory Pathologens Reference Laboratories for the investigation of the presence of C.diphtheriae, C.ulcerans and C.pseudotuberculosis. The growth in each plate were collected with a sterile swab and inoculated in tryptic soy broth. Following 2 hours of incubation at 37oC, subcultures were inoculated in cystine-tellurite-blood agar (CTBA) and 5% sheep-blood agar plates; after an overnight incubation tellurite-reducing colonies were inoculated in Tinsdale agar plates. The suspected colonies with positive cystinase activity were identified by conventional methods and also with Coryne API (Biomerieux, France) systems. Toxicity tests (ELEK, PCR) were performed to investigate whether the C.diphtheriae strains were producing toxins. A total of 500 patients were involved in the study. Of these 260 (52%) were girls and 240 (48%) were boys with a mean age of 76 (range, 21-213) months. All patients except one were fully vaccinated with boosters. Most common presenting symptoms of the patients were fever (19.8%), sore throat (52.6%), cough (49.2%), tonsillar hyperemia (97.6%), presence of crypt (24.6%), and membrane over tonsils (1%). Group A beta-hemolytic streptococcocci were detected in the throat swab cultures of 66 (%13.2) patients. Genotypically toxin negative C.diphtheriae biovar gravis was identified in the throat swab cultures of 3 patients (2 girls and 1 boy). The tonsils were hyperemic and hypertrophic in all the patients with C.diphtheriae biovar gravis. C.ulcerans and C.pseudotuberculosis were detected in none of the patients. It is considered that similar regular cross-sectional studies or routine screening programs are expected to raise awareness about this forgotten microorganism both epidemiologically and microbiologically.

Tularemia in children, Turkey, September 2009-November 2012.

Tularemia, a zoonotic disease caused by Francisella tularensis, is found throughout most of the Northern Hemisphere. It is not well known and is often misdiagnosed in children. Our aim with this study was to evaluate the diagnosis, treatment, and prognosis for 100 children with tularemia in Turkey. The mean patient age was 10.1 ± 3.5 years (range 3-18 years), and most (63%) patients were male. The most common physical signs and laboratory findings were cervical lymphadenopathy (92%) and elevated erythrocyte sedimentation rate (89%). Treatment response was higher and rate of relapse lower for children 5-10 years of age than for those in other age groups. Associated with treatment failure were female sex, treatment delay of ≥16 days, and use of doxycycline. Tularemia is endemic to Turkey, and the number of cases has been increasing among children as well as adults.

Water as Source of Francisella tularensis Infection in Humans, Turkey.

Francisella tularensis DNA extractions and isolates from the environment and humans were genetically characterized to elucidate environmental sources that cause human tularemia in Turkey. Extensive genetic diversity consistent with genotypes from human outbreaks was identified in environmental samples and confirmed water as a source of human tularemia in Turkey.

[Primary Neisseria meningitidis conjunctivitis in a 14-month-old child]. / On dört aylik bir çocukta primer Neisseria meningitidis konjonktiviti.

Meningococcal conjunctivitis is a rare but important infection since it can lead to severe complications and can threaten public health. It may emerge in two forms, either primary or secondary type which is developed after a systemic infection. Accurate diagnosis of primary meningococcal conjunctivitis is very important in addition to ocular complications which can result in loss of vision, the condition can also lead to severe complications like systemic meningococcal disease. However, the lack of specific symptoms which can distinguish meningococcal conjunctivitis from other forms of bacterial conjunctivitis, initiation of empiric antibiotic therapy without performing culture and nonaccurate differentiation of Neisseria gonorrhoeae and Neisseria meningitidis with commercial kits/systems used in laboratories cause problematic situations. This report describes a case of primary unilateral conjunctivitis in a 14-month-old girl caused by non-groupable N.meningitidis that was resolved without sequelae following treatment. A pre-healthy 14-month-old girl was brought to the pediatric emergency department with redness, crusts and discharge in the left eye that had begun two days earlier. Ocular examination revealed hyperemia and purulent discharge in the left conjunctiva. Purulent conjunctivitis was diagnosed. A conjunctival swab specimen was taken for culture, and the patient was started on topical netilmicin (4x1), topical fusidic acid (2x1) and artificial tears. Microscopic examination of the conjunctival swab revealed polymorphonuclear leukocytes and no visible bacteria. Catalase and oxidase positive, gram-negative diplococci grew purely in culture. The first Gram stain preparation was evaluated again after the growth and small numbers of gram-negative diplococci were observed. The cultivated bacteria were identified as N.meningitidis using MALDI-TOF MS (Bruker Daltonics, Germany), but as N.gonorrhoeae with BBL Crystal N/H (Neisseria/Haemophilus) (BD Diagnostic Systems, MD) identification system. The isolate was identified as N.meningitidis by polymerase chain reaction method. The isolate was sent to the Public Health Institution of Turkey for confirmation and serotyping. It was confirmed as non-groupable N.meningitidis. This is the first report of conjunctivitis caused by non-groupable N.meningitidis from Turkey. We wish to emphasize the importance of Gram staining and differentiation of the species by automatized systems in diagnosis, netilmicin may be one of the options for empiric treatment and in terms of public health the most appropriate approach may be evaluation of the severity of conjunctivitis and causative serogroup which depends on case-based approach.

[Seroprevalence of tularemia in risk groups of humans and animals in Van, eastern Turkey]. / Van ili ve çevresinde risk altindaki insan ve hayvan gruplarinda tularemi seroprevalansi.

Tularemia has become a re-emerging zoonotic disease in Turkey recently. The aims of this study were to determine the seroprevalence of tularemia in humans and their animals living in rural risky areas of our region and to investigate the risk factors. Between January and July 2012, people living in rural areas of Van province (located at eastern part of Turkey) and their domestic animals were included in the study. The sample size was determined by using cluster sampling method like in an event with known prevalence and planned as a cross-sectional epidemiological study. Proportional random sampling method was used to determine which individuals will be included in the study. Presence of tularemia antibodies in the sera of a total 495 voluntary persons (343 female, 152 male; age range: 18-79 years, mean age: 40.61) and their 171 animals (40 cattle, 124 sheep and 7 goats) were screened by microagglutination test using safranin O-stained F.tularensis antigen (Public Health Agency of Turkey). For the evaluation of cross-reactivity between Brucella spp., tularemia positive serum samples were also tested with brucella microagglutination test. Among human and animal samples, 11.9% (59/495) and 44% (76/171) yielded positive results with the titers of ≥ 1:20 in F.tularensis microagglutination test, respectively. However, 69.5% (41/59) of human sera and 78.9% (60/76) of animal sera demonstrated equal or higher titers in the brucella test, so those sera were considered as cross-reactive. After exclusion of these sera, the seroprevalence for F.tularensis were calculated as 3.6% (18/495) for humans and 9.4% (16/171) for animals. Among the 16 animals with positive results, 12 were sheep, three were cattle and one was goat. The difference between seropositivity rates among the domestic animal species was not statistically significant (p> 0.05). In addition, no statistically significant differences were found between risk factors including insect bite, tick bite, contact with rodents, eating the meat of hunted animals (rabbit), having pet (cat) in home (p> 0.05). In this study, the rate of tularemia seropositivity among humans was similar to the results of previous studies which were performed in our country; however the seropositivity rate of tularemia among domestic animals in our study was higher than the results of a few studies which were conducted on domestic animals. In conclusion, preventive procedures and precautions must be taken into consideration to control the transmission of the infection.

[Tularemia is spreading from north to south side of Turkey: a small outbreak in Kahramanmaras, Turkey]. / Tularemi Türkiye'nin kuzeyinden güneyine dogru yayiliyor: Kahramanmaras'da küçük bir salgin.

Tularemia is a zoonotic disease caused by Francisella tularensis. Sporadic tularemia cases have been increasingly reported particularly from provinces located at northwest and central regions of Turkey especially during last two decades, as well as waterborne outbreaks reported from almost all regions. Transmission most often occurs through consumption of contaminated water and food, thus, oropharyngeal form is the most common clinical presentation in our country. The aim of this study was to present a small outbreak experience in Afsin, country of Kahramanmaras province located at southern part of Turkey. A total of 10 patients (5 male, 5 female; age range 2-68 years; mean age 25 years) who were admitted to Afsin State Hospital with the complaints of swollen neck between 21 October 2013-22 January 2014, were evaluated considering their clinical findings and treatment outcomes. Following the diagnosis of the first tularemia case coming from Nadir village, a field investigation was performed. All villagers were informed about the disease and water samples from the possible sources of outbreak were collected by provincial health authorities. Lymph node aspirate and serum samples were sent for culture and serologic investigation and the environmental water samples were sent for molecular analysis to the National Tularemia Reference Laboratory at Public Health Institution of Turkey. Six out of 10 patients' sera were found positive in terms of F.tularensis antibodies between the titers of 1/320-1/1280 by microagglutination test (MAT) and diagnosis of oropharyngeal tularemia was based on the clinical and serological findings. One of the patients also presented with oculoglandular form accompanying oropharyngeal form. Cultures from aspirate samples that could be obtained from only two patients yielded negative results. Three out of six patients' lymph nodes were drained surgically and one was drained by ultrasound-guided needle. In one case lymph node suppuration occured spontaneously during examination. Three of the patients were treated with streptomycin, two with ciprofloxacin and one with doxycycline. Although no polymerase chain reaction positivities in terms of F.tularensis were detected in the water samples, the outbreak was thought to be waterborne since the hygienic conditions of the water sources were inadequate and the chlorination procedures were inappropriate. In conclusion, the detection of the first case of tularemia and the following small outbreak in Kahramanmaras region, emphasized that the disease has had a spreading trend from the endemic northern Anatolia to nonendemic southern provinces of Turkey.

[Evaluation of a newly-developed ready-to-use commercial PCR kit for the molecular diagnosis of Francisella tularensis]. / Francisella tularensis'in moleküler tanisinda yeni gelistirilen kullanima hazir ticari PCR kitinin etkinliginin degerlendirilmesi.

Tularemia is a rare zoonotic infection, however, considerations of tularemia as a biological weapon and several recent major epidemics have caused renewed interest in this disease. Laboratory diagnosis of tularemia is done in the presence of appropriate epidemiological data, by the demonstration of specific antibodies in the serum samples obtained with 1-2 week intervals following the development of symptoms. It is an a posteriori analysis with limited use for prompt diagnosis of the patient during the early symptomatic phase and deliberate release of biological agents. Limitations in both culture and serology have led to substantial research in the development of new diagnostic techniques. Several PCR methods for tularemia have been developed, both for conventional and real-time polymerase chain reaction (rtPCR). However, PCR methods are hard to be deployed in remote endemic areas that lack sufficient infrastructure. Recently a "Toolbox" which includes all instruments, equipments and solutions [DNA4U® Bacteria Genomic DNA Isolation Kit, CubeCycler® (Personal Thermal Cycler), PCR4U® Bioterrorism Agents Detection Kit, electrophoresis tank, power supply, ready-agarose gel and electrophoresis buffer] necessary for conventional PCR, was developed for the identification of bioterrorism agents in the field. In this study we aimed to evaluate the efficacy of a ready-to-use commercial PCR kit (Nanobiz, Ankara, Turkey) targeting the tul4 gene, for the diagnosis of tularemia and to compare the results with an in-house conventional PCR and a rtPCR test. We applied the assay to a collection of four F.tularensis standard strains, 15 field isolates (from humans, animals, water), 13 non-Francisella strains which are phylogenetically related to F.tularensis and a total of 60 lymph node aspirates obtained from suspected tularemia cases. Compared to the in-house PCR method used in our laboratory, the sensitivity, specificity, positive and negative predictive values of Nanobiz PCR Toolbox assay were found to be 100%. The lowest detection limit of this method was determined as 100 genomic equivalent per PCR reaction mix. The new PCR kit is a rapid and accurate alternative to the conventional PCR methods since the toolbox includes all of the required chemicals, accessories and equipments. This ready-to-use PCR assay was appraised to be a valuable diagnostic tool for the detection of F.tularensis in the outbreak settings particularly in remote areas with limited resources.